Re: [ccp4bb] Efficient way of showing residue conservation
Dear Bostjan, There is Chimera for almost anything you can think of. Search for Structure-Based Sequence Alignment on this page: http://www.cgl.ucsf.edu/chimera/features.html Petr On Dec 8, 2011, at 6:39 AM, Bostjan Kobe wrote: Consurf will do this for you. Bostjan --- Bostjan Kobe NHMRC Research Fellow Professor of Structural Biology School of Chemistry and Molecular Biosciences and Institute for Molecular Bioscience (Division of Chemistry and Structural Biology) and Centre for Infectious Disease Research Cooper Road University of Queensland Brisbane, Queensland 4072 Australia Phone: +61 7 3365 2132 Fax: +61 7 3365 4699 E-mail: b.k...@uq.edu.au URL: http://www.scmb.uq.edu.au/staff/bostjan-kobe Office: Building 76 Room 329 Notice: If you receive this e-mail by mistake, please notify me, and do not make any use of its contents. I do not waive any privilege, confidentiality or copyright associated with it. Unless stated otherwise, this e-mail represents only the views of the Sender and not the views of The University of Queensland. On 8/12/11 3:26 PM, Yuri Pompeu yuri.pom...@ufl.edu wrote: I once saw a figure showing the protein as surface, but instead of having it coloured by atom type or potential, it was shown by percent conservation in the family. Something like red highly conserved, all the way to white, not conserved at all... Now, I assume the figure was done by uploading aligned sequnces of several members of a family, and the colouring the generated surface accordingly. Does anyone know a way to do this more elegantly than what I tried doing? ps. I quit colouring them manually after I remebered my protein was 407 aa long...
Re: [ccp4bb] Efficient way of showing residue conservation
Do I feel stupid! My previous message about Chimera should have been addresses to Yuri Pompeu, but not to Bostjan Kobe. Sincerely, Petr On Dec 8, 2011, at 6:26 AM, Yuri Pompeu wrote: I once saw a figure showing the protein as surface, but instead of having it coloured by atom type or potential, it was shown by percent conservation in the family. Something like red highly conserved, all the way to white, not conserved at all... Now, I assume the figure was done by uploading aligned sequnces of several members of a family, and the colouring the generated surface accordingly. Does anyone know a way to do this more elegantly than what I tried doing? ps. I quit colouring them manually after I remebered my protein was 407 aa long...
Re: [ccp4bb] Efficient way of showing residue conservation
Just to add on others' tips: Consurf is interfaced to Chimera. Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Yuri Pompeu [yuri.pom...@ufl.edu] Sent: Thursday, December 08, 2011 7:26 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Efficient way of showing residue conservation I once saw a figure showing the protein as surface, but instead of having it coloured by atom type or potential, it was shown by percent conservation in the family. Something like red highly conserved, all the way to white, not conserved at all... Now, I assume the figure was done by uploading aligned sequnces of several members of a family, and the colouring the generated surface accordingly. Does anyone know a way to do this more elegantly than what I tried doing? ps. I quit colouring them manually after I remebered my protein was 407 aa long...
[ccp4bb] mmView - a tool for mmCIF exploration
Dear colleagues, We would like to announce the availability of mmView - the web-based application which allows to comfortably explore the structural data of biomacromolecules stored in the mmCIF (macromolecular Crystallographic Information File) format. The mmView software system is primarily intended for educational purposes but it can also serve as an auxiliary tool for working with biomolecular structures. The mmView application is offered in two flavors: as a publicly available web server http://ich.vscht.cz/projects/mmview/, and as an open-source stand-alone application (available from http://sourceforge.net/projects/mmview) that can be installed on the user’s computer. Petr Cech and Daniel Svozil -- Daniel Svozil, PhD Head of Laboratory of Informatics and Chemistry Institute of Chemical Technology Czech Republic phone: +420 220 444 391 http://ich.vscht.cz/~svozil
Re: [ccp4bb] Efficient way of showing residue conservation
8-Dec-2011 10:40am Dear Bostjan Consurf is also interfaced to Proteopedia, http://www.proteopedia.org, i.e. for all structures that have at least several sequences, one can automatically see an evolutionarily colored 3D Jmol image of the structures by just pushing the ConSurf button. See, e.g. http://proteopedia.org/wiki/index.php/1hho the STRUCTURE OF HUMAN OXYHAEMOGLOBIN AT 2.1 ANGSTROMS RESOLUTION by Boaz Shaanan and click on button Evolutionary conservation: [show] best regards, Joel On 8 Dec 2011, at 07:39, Bostjan Kobe wrote: Consurf will do this for you. Bostjan --- Bostjan Kobe NHMRC Research Fellow Professor of Structural Biology School of Chemistry and Molecular Biosciences and Institute for Molecular Bioscience (Division of Chemistry and Structural Biology) and Centre for Infectious Disease Research Cooper Road University of Queensland Brisbane, Queensland 4072 Australia Phone: +61 7 3365 2132 Fax: +61 7 3365 4699 E-mail: b.k...@uq.edu.aumailto:b.k...@uq.edu.au URL: http://www.scmb.uq.edu.au/staff/bostjan-kobe Office: Building 76 Room 329 Notice: If you receive this e-mail by mistake, please notify me, and do not make any use of its contents. I do not waive any privilege, confidentiality or copyright associated with it. Unless stated otherwise, this e-mail represents only the views of the Sender and not the views of The University of Queensland. On 8/12/11 3:26 PM, Yuri Pompeu yuri.pom...@ufl.edumailto:yuri.pom...@ufl.edu wrote: I once saw a figure showing the protein as surface, but instead of having it coloured by atom type or potential, it was shown by percent conservation in the family. Something like red highly conserved, all the way to white, not conserved at all... Now, I assume the figure was done by uploading aligned sequnces of several members of a family, and the colouring the generated surface accordingly. Does anyone know a way to do this more elegantly than what I tried doing? ps. I quit colouring them manually after I remebered my protein was 407 aa long...
Re: [ccp4bb] Efficient way of showing residue conservation
On 12/08/2011 05:11 PM, Petr Leiman wrote: Dear Bostjan, There is Chimera for almost anything you can think of. Not coot, you are sure? Because (i) Chimera is not part of CCP4 and (ii) usually coot does everything on this mailing list. :) Search for Structure-Based Sequence Alignment on this page: http://www.cgl.ucsf.edu/chimera/features.html Petr On Dec 8, 2011, at 6:39 AM, Bostjan Kobe wrote: Consurf will do this for you. Bostjan --- Bostjan Kobe NHMRC Research Fellow Professor of Structural Biology School of Chemistry and Molecular Biosciences and Institute for Molecular Bioscience (Division of Chemistry and Structural Biology) and Centre for Infectious Disease Research Cooper Road University of Queensland Brisbane, Queensland 4072 Australia Phone: +61 7 3365 2132 Fax: +61 7 3365 4699 E-mail: b.k...@uq.edu.au URL: http://www.scmb.uq.edu.au/staff/bostjan-kobe Office: Building 76 Room 329 Notice: If you receive this e-mail by mistake, please notify me, and do not make any use of its contents. I do not waive any privilege, confidentiality or copyright associated with it. Unless stated otherwise, this e-mail represents only the views of the Sender and not the views of The University of Queensland. On 8/12/11 3:26 PM, Yuri Pompeuyuri.pom...@ufl.edu wrote: I once saw a figure showing the protein as surface, but instead of having it coloured by atom type or potential, it was shown by percent conservation in the family. Something like red highly conserved, all the way to white, not conserved at all... Now, I assume the figure was done by uploading aligned sequnces of several members of a family, and the colouring the generated surface accordingly. Does anyone know a way to do this more elegantly than what I tried doing? ps. I quit colouring them manually after I remebered my protein was 407 aa long...
Re: [ccp4bb] Efficient way of showing residue conservation
I believe Charlie Bond's ALINE (http://crystal.bcs.uwa.edu.au/px/charlie/software/aline/) will let you make a nicely coloured sequence alignement, and then write out a Pymol script which will colour the surface by conservation. Mads -- Mads Gabrielsen, PhD Institute of Infection, Immunology and Inflammation College of Medical, Veterinary and Life Sciences University of Glasgow Room B216 /L303 GBRC 120 University place G12 8TA Phone: 0141 3307264 / 6180 E-mail: mads.gabriel...@glasgow.ac.uk On 08/12/2011 05:26, Yuri Pompeu yuri.pom...@ufl.edu wrote: I once saw a figure showing the protein as surface, but instead of having it coloured by atom type or potential, it was shown by percent conservation in the family. Something like red highly conserved, all the way to white, not conserved at all... Now, I assume the figure was done by uploading aligned sequnces of several members of a family, and the colouring the generated surface accordingly. Does anyone know a way to do this more elegantly than what I tried doing? ps. I quit colouring them manually after I remebered my protein was 407 aa long...
[ccp4bb] How to manually dock the rigid molecule into the active site
Dear All, I have a protein crystal co-crystallized with a rigid molecule (solved by small-molecule X-ray method). The color of that crystal is orange (native is colorless) after 2d cocrystallization experiment. Then I collected synchrotron data with a resolution at 1.25 Ang. I can solve it by MR. After I re-build slightly the protein and add some known ions/water, i found there are +ve connected blobs in the known active site of that protein. At first I suspect they are water/ion/EDO but many of them are partially connected within C-C/C-O distances (1.4-1.6 Ang) and in some part, i may imagine some broken six-membered ring, that are presumably found in that rigid molecule. How can I dock or confirm that rigid molecule using CCP4 software or COOT smartly? I tried to look at all density regions bit by bit, but the density for the protein atoms always interfere with my vision. Is it possible to mask out the density of protein atoms, leaving the residue density of interest for the rest atoms (maybe the rigid molecule)? many thanks in advance. stephen -- Dr. Stephen Sin-Yin Chui (徐先賢) Assistant Professor, Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China. Tel: 22415814 (Office), 22415818 (X-ray Diffraction Laboratory)
Re: [ccp4bb] Efficient way of showing residue conservation-thank you everyone
On Thu, 8 Dec 2011 09:19:57 +, Mads Gabrielsen wrote: I believe Charlie Bond's ALINE (http://crystal.bcs.uwa.edu.au/px/charlie/software/aline/) will let you make a nicely coloured sequence alignement, and then write out a Pymol script which will colour the surface by conservation. Mads -- Mads Gabrielsen, PhD Institute of Infection, Immunology and Inflammation College of Medical, Veterinary and Life Sciences University of Glasgow Room B216 /L303 GBRC 120 University place G12 8TA Phone: 0141 3307264 / 6180 E-mail: mads.gabriel...@glasgow.ac.uk On 08/12/2011 05:26, Yuri Pompeu yuri.pom...@ufl.edu wrote: I once saw a figure showing the protein as surface, but instead of having it coloured by atom type or potential, it was shown by percent conservation in the family. Something like red highly conserved, all the way to white, not conserved at all... Now, I assume the figure was done by uploading aligned sequnces of several members of a family, and the colouring the generated surface accordingly. Does anyone know a way to do this more elegantly than what I tried doing? ps. I quit colouring them manually after I remebered my protein was 407 aa long... -- Yuri Pompeu
Re: [ccp4bb] How to manually dock the rigid molecule into the active site
On Thu, 2011-12-08 at 17:49 +0800, Dr. STEPHEN SIN-YIN, CHUI wrote: I tried to look at all density regions bit by bit, but the density for the protein atoms always interfere with my vision. Is it possible to mask out the density of protein atoms, Isn't that what difference density map is for? Usually one simply generates the ligand model by using prodrg and/or sketcher (which would give you a monomer description) and then tries to manually dock it into the density in coot. Some automation is available (e.g. coot can search for a ligand in the difference map throughout the cell), but I believe it does not substitute for the manual inspection (and, imho, neither is such complete replacement possible or even desirable). Things appear to be tricky in your case - at 1.25A a well ordered ligand should produce clearly interpretable density. What you are describing sounds like either disordered ligand or indeed solvent molecules, or the mix of the two (the worst-case scenario). Ultimately, it is up to you to interpret the density, but be careful to curb your imagination. You may be able to get a better advice if you post the density snapshots. Cheers, Ed. -- Hurry up before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] better way to post your density snapshots
posting these attachments as links rather than attachments in CCP4bb messages is the way to go I think. many institutions offer this service (ours does), and there are also free and for-pay online ways to do this (for example www.yousendit.com, but there are many others). Then it is also not a problem to send (sorry, link) even large files. The only disadvantage I can think of is that they expire after some time. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij On 8 Dec 2011, at 16:39, Ed Pozharski wrote: Colleagues, One recurring question on this bb is I got this blob of density - is it my ligand or what in the name of pink unicorns this is? Often, a screen snapshot is posted, which is very helpful. But it may be better if those helping out could rotate density around in 3D. Understandably, posting the full model/map is not the way to go. However, I'd see no harm in posting just a small cutout of the map in the region of interest. It's not a difficult task (fft/mapmask or perhaps some usf magic), but is there some user-friendly approach to cutting out a small map volume? One can use coot to mask the map and then export it, but this seems to generate the ccp4-formatted map that covers more than just the masked region, thus the files are fairly large. Does anyone know of a simple solution other than placing dummy atoms in the region of interest and running fft/mapmask combination? (Is there phenix.cut_the_map_around_this_weird_blob ? :) Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] better way to post your density snapshots
I am not sure that sending links is the way to go. Our company blocks many websites that it considers not of professionel interest and were employees may spend too much time. More often than not I find that I cannot open the link provided to the bullitin board. E.g. I just checked the yousendit site and it is blocked. I think what Ed proposed, a map of maybe 10 Å**3 plus a pdb file with only a few residues would be better. Also properly compressed pictures (jpeg) are not that big that they cannot be sent by email. Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mark J van Raaij Sent: Thursday, December 08, 2011 4:50 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] better way to post your density snapshots posting these attachments as links rather than attachments in CCP4bb messages is the way to go I think. many institutions offer this service (ours does), and there are also free and for-pay online ways to do this (for example www.yousendit.com, but there are many others). Then it is also not a problem to send (sorry, link) even large files. The only disadvantage I can think of is that they expire after some time. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij On 8 Dec 2011, at 16:39, Ed Pozharski wrote: Colleagues, One recurring question on this bb is I got this blob of density - is it my ligand or what in the name of pink unicorns this is? Often, a screen snapshot is posted, which is very helpful. But it may be better if those helping out could rotate density around in 3D. Understandably, posting the full model/map is not the way to go. However, I'd see no harm in posting just a small cutout of the map in the region of interest. It's not a difficult task (fft/mapmask or perhaps some usf magic), but is there some user-friendly approach to cutting out a small map volume? One can use coot to mask the map and then export it, but this seems to generate the ccp4-formatted map that covers more than just the masked region, thus the files are fairly large. Does anyone know of a simple solution other than placing dummy atoms in the region of interest and running fft/mapmask combination? (Is there phenix.cut_the_map_around_this_weird_blob ? :) Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] adxv
Hi Stacy, If you give the CCP4 program crank a try, I can give you advice, if needed. It can work with SAD data. Best wishes, Raj On Thu, 8 Dec 2011, stacy William wrote: Dear All...I am new user for Phenix. I am right now working with SAD data. I had run Ausol in GUI interface with my SAD anamolous data. but when i am using the output file for my autobuild program it is giving problems with Resolve... n terminating with an error message suggesting to check resolve file. Do i need to change some parameters in Autosol to produce right output files which in turn can work as input files for Autobuild program Thanks Stacy On Tue, Nov 22, 2011 at 8:21 PM, David Waterman dgwater...@gmail.com wrote: In addition, perhaps a safer test of ADXV than the set of Mar IP frames I suggested is by using a file from an ADSC detector, such as this one (thanks to James Holton): http://bl831.als.lbl.gov/example_data_sets/ALS/831/Gd_lyso1/ALS831_lyso_Gd_ 001.img I have no reason to believe the image plate frames shouldn't work with ADXV other than that I haven't tested it. I would be extremely surprised however if the ADSC image doesn't work with a properly functioning ADXV. -- David On 22 November 2011 14:41, Andrew Purkiss a.purk...@mail.cryst.bbk.ac.uk wrote: Hi Rajesh, That is a very old version (1.9.4) of ADXV. Try the current version from here http://www.scripps.edu/~arvai/adxv/ version 1.9.8 works fine for us and opens Pilatus data without modifying the files. Hope this helps. Andrew Purkiss -- X-ray Laboratory Manager, London Research Institute, Cancer Research UK. On Tue, 2011-11-22 at 09:30 -0500, Rajesh kumar wrote: Hi Andrzej, I used exactly what you have suggested. Downloaded file adxv.i686FC2.tgz from some place. untar Gunzip so I get a folder adxv.1686FC2 which has adxv and adxv.1686FC2_static files. I made both executable using chmod. I put the PATH in .bashrc If I run this command in the folder where images are then it opens with Windows open but the file selection dialogue is locked and the selection pattern is 0.*. But not functional and get the error I posted recently pin8]$ adxv pin8_1_144.img (standard_in) 1: parse error (standard_in) 1: parse error (standard_in) 1: parse error beam_center x = pixels , mm beam_center y = pixels , mm distance = mm overload = counts pixelsize = 0.172 mm wavelength = Angstroem Adxv Version 1.9.4-beta Copyright (C) 1994-2007 by Andrew Arvai, Area Detector Systems Corporation Using 24-bit TrueColor visual Warning: translation table syntax error: Unknown keysym name: osfActivate Warning: ... found while parsing ':KeyosfActivate: ManagerParentActivate()' Warning: String to TranslationTable conversion encountered errors Warning: translation table syntax error: Unknown keysym name: osfBeginLine Warning: ... found while parsing ':KeyosfBeginLine: ManagerGadgetTraverseHome()' Warning: String to TranslationTable conversion encountered errors Warning: translation table syntax error: Unknown keysym name: osfHelp Warning: ... found while parsing ':KeyosfHelp: ManagerGadgetHelp()' Warning: String to TranslationTable conversion encountered errors Warning: translation table syntax error: Unknown keysym name: osfActivate Warning: ... found while parsing ':KeyosfActivate: ManagerParentActivate()' Warning: String to TranslationTable conversion encountered errors Warning: translation table syntax error: Unknown keysym name: osfActivate Warning: ... found while parsing ':KeyosfActivate: PrimitiveParentActivate()' Warning: String to TranslationTable conversion encountered errors Warning: translation table syntax error: Unknown keysym name: osfHelp Warning: ... found while parsing ':KeyosfHelp: Help()' Warning: String to TranslationTable conversion encountered errors Warning: translation table syntax error: Unknown keysym name: osfActivate Warning: ... found while parsing ':KeyosfActivate: PrimitiveParentActivate()' Warning: String to TranslationTable conversion encountered errors Warning: translation table syntax error: Unknown keysym name: osfCancel Warning: ... found while parsing ':KeyosfCancel: MenuEscape()' Warning: String to TranslationTable conversion encountered errors Warning: translation table syntax error: Unknown
Re: [ccp4bb] better way to post your density snapshots
herman.schreu...@sanofi.com wrote: I am not sure that sending links is the way to go. Our company blocks many websites that it considers not of professionel interest and were employees may spend too much time. What a brilliant idea! If only this could be implemented in academia. Our Ph.D. students would stop asking stupid questions on this BB but instead read books and journal articles. Yes, everything except PDB, sciencedirect, webofscience, pubmed and arxiv (this one is for real science geeks) must be blocked! Petr -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mark J van Raaij Sent: Thursday, December 08, 2011 4:50 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] better way to post your density snapshots posting these attachments as links rather than attachments in CCP4bb messages is the way to go I think. many institutions offer this service (ours does), and there are also free and for-pay online ways to do this (for example www.yousendit.com, but there are many others). Then it is also not a problem to send (sorry, link) even large files. The only disadvantage I can think of is that they expire after some time. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij On 8 Dec 2011, at 16:39, Ed Pozharski wrote: Colleagues, One recurring question on this bb is I got this blob of density - is it my ligand or what in the name of pink unicorns this is? Often, a screen snapshot is posted, which is very helpful. But it may be better if those helping out could rotate density around in 3D. Understandably, posting the full model/map is not the way to go. However, I'd see no harm in posting just a small cutout of the map in the region of interest. It's not a difficult task (fft/mapmask or perhaps some usf magic), but is there some user-friendly approach to cutting out a small map volume? One can use coot to mask the map and then export it, but this seems to generate the ccp4-formatted map that covers more than just the masked region, thus the files are fairly large. Does anyone know of a simple solution other than placing dummy atoms in the region of interest and running fft/mapmask combination? (Is there phenix.cut_the_map_around_this_weird_blob ? :) Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] model building at 3.2 A
On the other hand, shooting a lower resolution crystal may get you the conformation of the disordered domain. Surprising at first thought, but was true in p97/VCP from the Brunger Lab. F On Dec 7, 2011, at 11:47 PM, atul kumar wrote: Dear all I have crytals which diffract up to 3.2 A at synchrotron, I am solving this by molecular replacement. I have built 70% of the model successfully,but the problem is it have a very poor density for some 30-40 residues at N terminal. I can't build anything in this region,this could be because of disordered structure or because of low resolution.what are the things which I can try to improve this? suggestion are requested! thanks regards' Atul Kumar
Re: [ccp4bb] model building at 3.2 A
Hmm- I wonder if B-factor unsharpening (applying a large POSITIVE B-factor to the data) would have the same effect? Francis E Reyes wrote: On the other hand, shooting a lower resolution crystal may get you the conformation of the disordered domain. Surprising at first thought, but was true in p97/VCP from the Brunger Lab. F On Dec 7, 2011, at 11:47 PM, atul kumar wrote: Dear all I have crytals which diffract up to 3.2 A at synchrotron, I am solving this by molecular replacement. I have built 70% of the model successfully,but the problem is it have a very poor density for some 30-40 residues at N terminal. I can't build anything in this region,this could be because of disordered structure or because of low resolution.what are the things which I can try to improve this? suggestion are requested! thanks regards' Atul Kumar
Re: [ccp4bb] model building at 3.2 A
On Thu, 2011-12-08 at 12:17 +0530, atul kumar wrote: I can't build anything in this region,this could be because of disordered structure or because of low resolution. Both, and also the presence of disordered fragment may be the reason the resolution is low, thus the already mentioned suggestion to remove it (unless, of course, that renders your protein inactive and thus compromises your structure-based conclusions). what are the things which I can try to improve this? If the region in question is disordered in solution, pretty much nothing. Look for a different crystal form. Having a disordered region does not make your structure invalid. In fact, it may be important for understanding the biological role of the protein. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] better way to post your density snapshots
There is indeed the phenix.cut_out_density tool (by Tom Terwilliger) which does a nice job of reducing the size of the output mtz-file (it shifts the cutout region to the origin and reduces the unit cell). On the link-versus-attachment issue, certainly the link is preferred, but the aforementioned tool produced in my tests the mtz-file as small as 50kb, which is not too bad. -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs
Re: [ccp4bb] better way to post your density snapshots
For the second time today I have to apologize. In no way I wanted to discourage anyone and especially people new to crystallography from posting questions to this BB. Especially good thoughtful questions. Sincerely, Petr On Dec 8, 2011, at 5:24 PM, Petr Leiman wrote: herman.schreu...@sanofi.com wrote: I am not sure that sending links is the way to go. Our company blocks many websites that it considers not of professionel interest and were employees may spend too much time. What a brilliant idea! If only this could be implemented in academia. Our Ph.D. students would stop asking stupid questions on this BB but instead read books and journal articles. Yes, everything except PDB, sciencedirect, webofscience, pubmed and arxiv (this one is for real science geeks) must be blocked! Petr -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mark J van Raaij Sent: Thursday, December 08, 2011 4:50 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] better way to post your density snapshots posting these attachments as links rather than attachments in CCP4bb messages is the way to go I think. many institutions offer this service (ours does), and there are also free and for-pay online ways to do this (for example www.yousendit.com, but there are many others). Then it is also not a problem to send (sorry, link) even large files. The only disadvantage I can think of is that they expire after some time. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij On 8 Dec 2011, at 16:39, Ed Pozharski wrote: Colleagues, One recurring question on this bb is I got this blob of density - is it my ligand or what in the name of pink unicorns this is? Often, a screen snapshot is posted, which is very helpful. But it may be better if those helping out could rotate density around in 3D. Understandably, posting the full model/map is not the way to go. However, I'd see no harm in posting just a small cutout of the map in the region of interest. It's not a difficult task (fft/mapmask or perhaps some usf magic), but is there some user-friendly approach to cutting out a small map volume? One can use coot to mask the map and then export it, but this seems to generate the ccp4-formatted map that covers more than just the masked region, thus the files are fairly large. Does anyone know of a simple solution other than placing dummy atoms in the region of interest and running fft/mapmask combination? (Is there phenix.cut_the_map_around_this_weird_blob ? :) Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] model building at 3.2 A
Dear Kumar Could you please provide some information on your refinement protocol/progress and quality of your partial model thus far. Also, what is the quality and completeness of your data, in particular in the lowest resolution to 10 angs range. In this way one might be able to provide more concrete feedback. Experience from several structures around such resolution tells me that it is very much worth spending time optimizing what you can reliably fit and refine. This approach will pay off handsomely in terms of map quality and might lead to modeling what now may seem uninterpretable. The latest Phenix and Refmac versions are great, and their B-sharpening protocols can reveal a lot. In addition Buster with its handling of missing domains in Fc can also help a lot. Did you try any phase improvement by density modification? No. of molecules in asu? Also getting some experimental phase info (even at low resolution) might also help. Best regards Savvas On 08 Dec 2011, at 07:47, atul kumar atulsingh21...@gmail.com wrote: Dear all I have crytals which diffract up to 3.2 A at synchrotron, I am solving this by molecular replacement. I have built 70% of the model successfully,but the problem is it have a very poor density for some 30-40 residues at N terminal. I can't build anything in this region,this could be because of disordered structure or because of low resolution.what are the things which I can try to improve this? suggestion are requested! thanks regards' Atul Kumar
[ccp4bb] PHENIX vs REFMAC refinement had me fooled
Dear All, Question: Has anybody ever refined the same structure using PHENIX and then tried REFMAC to see what happens? I did and I stumbled on something funny. I'm refining a structure at 1.1A resolution which was solved with Iodine phasing using PHENIX AutoSolve. Got a great map and the structure was built almost completely. I had to build a few residues myself, and using the published sequence, I started filling in the residues, but as I came nearer the N-terminus, it looked like the density did not match residues from the sequence. I kept the residues as in the sequence, but as you can see from the PHENIX refined picture (below is the link) it still looks like the amino acid sequence in the crystal does not match the published protein sequence. Out of interest I refined the same file in REFMAC, and now the electron density is correct, and the sequence of the amino acids in the crystal matches the published sequence (see link for picture below). Not only that. my R/Rfree improved (16.5/19 for PHENIX, 10/18 for REFMAC). I've also refined the occupancies of the iodide, however the the output FO-FC map from PHENIX complains and the REFMAC map is fine. How can this be and what causes this? Link for the pictures: Both maps are at identical Sigma levels in both pictures. PHENIX: http://dl.dropbox.com/u/51868657/PHENIX_refined.png REFMAC: http://dl.dropbox.com/u/51868657/REFMAC_refined.png Cheers, Chris Browning -- Dr. Christopher Browning Post-Doctor to Prof. Petr Leiman EPFL BSP-416 1015 Lausanne Switzerland Tel: 0041 (0) 02 16 93 04 40
Re: [ccp4bb] PHENIX vs REFMAC refinement had me fooled
Christopher, if you send me the input PDB and data files (off-list, of course) I will have a close look and then explain what exactly happens and why. I also promise to post the summary on this mailing list (without revealing the identity of your structure, of course). If you send me the command you used to run PHENIX, I will comment on that too. Please send the files to my email address: pafon...@lbl.gov Pavel On Thu, Dec 8, 2011 at 9:36 AM, Christopher Browning christopher.brown...@epfl.ch wrote: Dear All, Question: Has anybody ever refined the same structure using PHENIX and then tried REFMAC to see what happens? I did and I stumbled on something funny. I'm refining a structure at 1.1A resolution which was solved with Iodine phasing using PHENIX AutoSolve. Got a great map and the structure was built almost completely. I had to build a few residues myself, and using the published sequence, I started filling in the residues, but as I came nearer the N-terminus, it looked like the density did not match residues from the sequence. I kept the residues as in the sequence, but as you can see from the PHENIX refined picture (below is the link) it still looks like the amino acid sequence in the crystal does not match the published protein sequence. Out of interest I refined the same file in REFMAC, and now the electron density is correct, and the sequence of the amino acids in the crystal matches the published sequence (see link for picture below). Not only that. my R/Rfree improved (16.5/19 for PHENIX, 10/18 for REFMAC). I've also refined the occupancies of the iodide, however the the output FO-FC map from PHENIX complains and the REFMAC map is fine. How can this be and what causes this? Link for the pictures: Both maps are at identical Sigma levels in both pictures. PHENIX: http://dl.dropbox.com/u/51868657/PHENIX_refined.png REFMAC: http://dl.dropbox.com/u/51868657/REFMAC_refined.png Cheers, Chris Browning -- Dr. Christopher Browning Post-Doctor to Prof. Petr Leiman EPFL BSP-416 1015 Lausanne Switzerland Tel: 0041 (0) 02 16 93 04 40
Re: [ccp4bb] PHENIX vs REFMAC refinement had me fooled
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Christopher, if your R/Rfree from Refmac5 really are 10% vs. 18%, you might simply be looking at an electron density map with strong model bias, i.e. the map shows the features of the model and not of the data. Although at 1.1A resolution this seems quite unlikely, but that's what might explain this great gap between R and Rfree. Tim On 12/08/2011 06:36 PM, Christopher Browning wrote: Dear All, Question: Has anybody ever refined the same structure using PHENIX and then tried REFMAC to see what happens? I did and I stumbled on something funny. I'm refining a structure at 1.1A resolution which was solved with Iodine phasing using PHENIX AutoSolve. Got a great map and the structure was built almost completely. I had to build a few residues myself, and using the published sequence, I started filling in the residues, but as I came nearer the N-terminus, it looked like the density did not match residues from the sequence. I kept the residues as in the sequence, but as you can see from the PHENIX refined picture (below is the link) it still looks like the amino acid sequence in the crystal does not match the published protein sequence. Out of interest I refined the same file in REFMAC, and now the electron density is correct, and the sequence of the amino acids in the crystal matches the published sequence (see link for picture below). Not only that. my R/Rfree improved (16.5/19 for PHENIX, 10/18 for REFMAC). I've also refined the occupancies of the iodide, however the the output FO-FC map from PHENIX complains and the REFMAC map is fine. How can this be and what causes this? Link for the pictures: Both maps are at identical Sigma levels in both pictures. PHENIX: http://dl.dropbox.com/u/51868657/PHENIX_refined.png REFMAC: http://dl.dropbox.com/u/51868657/REFMAC_refined.png Cheers, Chris Browning - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFO4PjgUxlJ7aRr7hoRAszXAKCNmTZvCVaDUm6v3lQjp051H+ilDgCgxd1l as9CcWEseq9uEV8qMZsOfsg= =KKUr -END PGP SIGNATURE-
Re: [ccp4bb] PHENIX vs REFMAC refinement had me fooled
are you sure that you are using the original input intensities for the REFMAC map calculation (and the refinement run) and not the output ones of the (previous) run? if you are not, you may have model bias, and Rfree can be fooled, especially with NCS (if you have it). - or perhaps anisotropic B-factor refinement (if you are using it), works better in REFMAC than PHENIX? Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij On 8 Dec 2011, at 18:36, Christopher Browning wrote: Dear All, Question: Has anybody ever refined the same structure using PHENIX and then tried REFMAC to see what happens? I did and I stumbled on something funny. I'm refining a structure at 1.1A resolution which was solved with Iodine phasing using PHENIX AutoSolve. Got a great map and the structure was built almost completely. I had to build a few residues myself, and using the published sequence, I started filling in the residues, but as I came nearer the N-terminus, it looked like the density did not match residues from the sequence. I kept the residues as in the sequence, but as you can see from the PHENIX refined picture (below is the link) it still looks like the amino acid sequence in the crystal does not match the published protein sequence. Out of interest I refined the same file in REFMAC, and now the electron density is correct, and the sequence of the amino acids in the crystal matches the published sequence (see link for picture below). Not only that. my R/Rfree improved (16.5/19 for PHENIX, 10/18 for REFMAC). I've also refined the occupancies of the iodide, however the the output FO-FC map from PHENIX complains and the REFMAC map is fine. How can this be and what causes this? Link for the pictures: Both maps are at identical Sigma levels in both pictures. PHENIX: http://dl.dropbox.com/u/51868657/PHENIX_refined.png REFMAC: http://dl.dropbox.com/u/51868657/REFMAC_refined.png Cheers, Chris Browning -- Dr. Christopher Browning Post-Doctor to Prof. Petr Leiman EPFL BSP-416 1015 Lausanne Switzerland Tel: 0041 (0) 02 16 93 04 40
Re: [ccp4bb] better way to post your density snapshots
On Dec 8, 2011, at 8:39 AM, Ed Pozharski wrote:However, I'd see noharm in posting just a small cutout of the map in the region ofinterest. It's not a difficult task (fft/mapmask or perhaps some usfmagic), but is there some user-friendly approach to cutting out a smallmap volume?There's probably a slick program somewhere, but attached is a command line tool I made several years ago and still use all of the time. To use it, you need ccp4 and mapman from USF in your path. Running the script with "mapreg -h" yields the documentation below. It can cut a map using a box, extend or cut to the ASU, extend a map to a cell, or use a pdb file to make a box. It can also cull a map around a PDB.To use it, the attachment needs to be untarred (tar zxvf mapreg.tar).One of these days I may give it a web page.James* mapreg version 0.02*** output a region of a cns map*** copyright James C. Stroud, 2008*** distributed under the GNU Public License**Usage: mapreg [-h] [-c weight] [-b border] [-s symm] [-t type] [-x 'a b c alpha beta gamma'] mapfile [x1 y1 z1 x2 y2 z2 | ASU | CELL | pdbfile ]Flags: -h print this help -b border define a border if using a pdb file to define region default is 5 (5 Angstroms) -c weight culls according to a weighting factor between 0 and 1 -x cell the sides and angles *must* be in single quotes -s symm usually symmetry number is needed (ispcgr number) -t type type of input map - default is cns -o outfile name of output file (name generated if not supplied)Description: Mapreg takes a cns map as input and outputs a new region of the map as specified at the command line. The possible region specifiers are: x1 y1 z1 x2 y2 z2 : the region defined by the two grid unit or fractional coordinate points (x1,y1,z1) and (x2,y2,z2) 'ASU' : the CCP4 default asymmetric unit 'CELL' : the whole unit cell pdbfile : a pdb file defining the limits of the regionif border is defined, then this will be the borderin Angstroms around pdbfile to define the region If the region specifier is left out, then mapreg will output the whole unit cell. CULLING === If a culling factor is supplied and a pdb file is used for trimming, then culling will be attempted. Culling trims the map to the the atoms of the pdb file if supplied. Without the pdb file, the program terminates with an error if a culling factor is supplied. The culling weighting factor is, for all practical purposes, arbitrary. Play with it to get the desired results. Start with 0.1 and go up (tighter) or down (less tight). Make sure you are aware of the caveats of culling before you use this to make figures for publication. mapreg.tar Description: Unix tar archive
[ccp4bb] [ANNOUNCEMENT] mapreg has a home page
Hello all, Since no one offered a good utility for Ed Pozharski's idea to easily cut a region of a density map, I made a permanent web location for my utility called mapreg: http://mapreg.bravais.net/ Enjoy. James
[ccp4bb] The stupid/naive question
Hello folks, After a discussion with a colleague a question aroused regarding precipitants in crystallisation conditions. I must confess that I do not know if it is a really naive question or just a stupid one (guess the second option thought...), anyhow there we go: Imagine you have crystallisation condition that is more or less successful but not so great, that is, you are looking for some optimisation. Your condition let say is 5% PEG 20K. Assuming a world where relationships are linear (and I know this is not true, by the way...), I would say that I can change 5%-PEG20K by 50%-PEG2K. So the question is, does anyone know if someone has invest some time in discovering this kind of relationships between main precipitants used in macromolecular crystallography? The reason for this kind of weird question is that currently one of our cryo-protection protocol really damage our crystals and I was wondering if the same crystal could grow in a cryo-condition or something closer to it. Thank you for investing some time in such a awkward question!!! -- Israel Sanchez Fernandez PhD Ramakrishnan-lab MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK
Re: [ccp4bb] PHENIX vs REFMAC refinement had me fooled
In addition to the remarks of Mark and Tim, could you make sure that you are refining in Refmac with the correct flag for the Rfree set (value = 0)? In Phenix, this is the opposite: the value is 1 for test reflections and 0 for work reflections. So going from a PHENIX environment to Refmac, you might well be refining against your small fraction of test reflections. I have seen this give spurious density features (obviously) and serious model bias. Cheers Jonathan Ghent University On 08 Dec 2011, at 18:50, Mark J van Raaij wrote: are you sure that you are using the original input intensities for the REFMAC map calculation (and the refinement run) and not the output ones of the (previous) run? if you are not, you may have model bias, and Rfree can be fooled, especially with NCS (if you have it). - or perhaps anisotropic B-factor refinement (if you are using it), works better in REFMAC than PHENIX? Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij On 8 Dec 2011, at 18:36, Christopher Browning wrote: Dear All, Question: Has anybody ever refined the same structure using PHENIX and then tried REFMAC to see what happens? I did and I stumbled on something funny. I'm refining a structure at 1.1A resolution which was solved with Iodine phasing using PHENIX AutoSolve. Got a great map and the structure was built almost completely. I had to build a few residues myself, and using the published sequence, I started filling in the residues, but as I came nearer the N-terminus, it looked like the density did not match residues from the sequence. I kept the residues as in the sequence, but as you can see from the PHENIX refined picture (below is the link) it still looks like the amino acid sequence in the crystal does not match the published protein sequence. Out of interest I refined the same file in REFMAC, and now the electron density is correct, and the sequence of the amino acids in the crystal matches the published sequence (see link for picture below). Not only that. my R/Rfree improved (16.5/19 for PHENIX, 10/18 for REFMAC). I've also refined the occupancies of the iodide, however the the output FO-FC map from PHENIX complains and the REFMAC map is fine. How can this be and what causes this? Link for the pictures: Both maps are at identical Sigma levels in both pictures. PHENIX: http://dl.dropbox.com/u/51868657/PHENIX_refined.png REFMAC: http://dl.dropbox.com/u/51868657/REFMAC_refined.png Cheers, Chris Browning -- Dr. Christopher Browning Post-Doctor to Prof. Petr Leiman EPFL BSP-416 1015 Lausanne Switzerland Tel: 0041 (0) 02 16 93 04 40
Re: [ccp4bb] PHENIX vs REFMAC refinement had me fooled
Dear Tim, I agree with you completely. The question then becomes why does the automatic weighting scheme in refmac allow R and R-free to run away from each other by 8% in a 1.1 A resolution structure? Petr On Dec 8, 2011, at 6:50 PM, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Christopher, if your R/Rfree from Refmac5 really are 10% vs. 18%, you might simply be looking at an electron density map with strong model bias, i.e. the map shows the features of the model and not of the data. Although at 1.1A resolution this seems quite unlikely, but that's what might explain this great gap between R and Rfree. Tim On 12/08/2011 06:36 PM, Christopher Browning wrote: Dear All, Question: Has anybody ever refined the same structure using PHENIX and then tried REFMAC to see what happens? I did and I stumbled on something funny. I'm refining a structure at 1.1A resolution which was solved with Iodine phasing using PHENIX AutoSolve. Got a great map and the structure was built almost completely. I had to build a few residues myself, and using the published sequence, I started filling in the residues, but as I came nearer the N-terminus, it looked like the density did not match residues from the sequence. I kept the residues as in the sequence, but as you can see from the PHENIX refined picture (below is the link) it still looks like the amino acid sequence in the crystal does not match the published protein sequence. Out of interest I refined the same file in REFMAC, and now the electron density is correct, and the sequence of the amino acids in the crystal matches the published sequence (see link for picture below). Not only that. my R/Rfree improved (16.5/19 for PHENIX, 10/18 for REFMAC). I've also refined the occupancies of the iodide, however the the output FO-FC map from PHENIX complains and the REFMAC map is fine. How can this be and what causes this? Link for the pictures: Both maps are at identical Sigma levels in both pictures. PHENIX: http://dl.dropbox.com/u/51868657/PHENIX_refined.png REFMAC: http://dl.dropbox.com/u/51868657/REFMAC_refined.png Cheers, Chris Browning - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFO4PjgUxlJ7aRr7hoRAszXAKCNmTZvCVaDUm6v3lQjp051H+ilDgCgxd1l as9CcWEseq9uEV8qMZsOfsg= =KKUr -END PGP SIGNATURE-
Re: [ccp4bb] The stupid/naive question
That's a good question ! Here is my take on it: We are talking of say 5% PEG 2K weight per weight. So 5% PEG 2K and 5% PEG 20K contains ~ the same weight of polymer per liter of solution, and therefore the ~ same molarity of the ethylene oxide motif. Hence neglecting the effect of the ends of the polymer one would expect 5% PEG 2K and 5% PEG 20K to be nearly equivalent. In practice there are some variations in the required PEG concentration, but it certainly does not vary say 5-fold between PEG 1K and PEG 5K. Also the following is often true: small PEGs 800 trend all the behave the same, but as a different class of precipitant than medium-sized PEG, which themselves are different than the very large PEG (10K and above). So if a protein crystallizes with PEG 4K as precipitant it *usually* does not crystallize with PEG 400, and the same can be said for PEG 20K vs. PEG 2K. The small PEGs are much better cryoprotectants. Thierry From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Israel Sanchez Sent: Thursday, December 08, 2011 2:40 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] The stupid/naive question Hello folks, After a discussion with a colleague a question aroused regarding precipitants in crystallisation conditions. I must confess that I do not know if it is a really naive question or just a stupid one (guess the second option thought...), anyhow there we go: Imagine you have crystallisation condition that is more or less successful but not so great, that is, you are looking for some optimisation. Your condition let say is 5% PEG 20K. Assuming a world where relationships are linear (and I know this is not true, by the way...), I would say that I can change 5%-PEG20K by 50%-PEG2K. So the question is, does anyone know if someone has invest some time in discovering this kind of relationships between main precipitants used in macromolecular crystallography? The reason for this kind of weird question is that currently one of our cryo-protection protocol really damage our crystals and I was wondering if the same crystal could grow in a cryo-condition or something closer to it. Thank you for investing some time in such a awkward question!!! -- Israel Sanchez Fernandez PhD Ramakrishnan-lab MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] PHENIX vs REFMAC refinement had me fooled
In a non-computational capacity would also suggest perhaps resequencing your clone. Occasionally the published sequences are off, the specific base is polymorphous or there is also the possibility that you introduced a mutation somewhere. That would be the cheap and easy way to definitively answer the question. Cheers, Katherine On Thu, Dec 8, 2011 at 1:43 PM, Petr Leiman petr.lei...@epfl.ch wrote: Dear Tim, I agree with you completely. The question then becomes why does the automatic weighting scheme in refmac allow R and R-free to run away from each other by 8% in a 1.1 A resolution structure? Petr On Dec 8, 2011, at 6:50 PM, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Christopher, if your R/Rfree from Refmac5 really are 10% vs. 18%, you might simply be looking at an electron density map with strong model bias, i.e. the map shows the features of the model and not of the data. Although at 1.1A resolution this seems quite unlikely, but that's what might explain this great gap between R and Rfree. Tim On 12/08/2011 06:36 PM, Christopher Browning wrote: Dear All, Question: Has anybody ever refined the same structure using PHENIX and then tried REFMAC to see what happens? I did and I stumbled on something funny. I'm refining a structure at 1.1A resolution which was solved with Iodine phasing using PHENIX AutoSolve. Got a great map and the structure was built almost completely. I had to build a few residues myself, and using the published sequence, I started filling in the residues, but as I came nearer the N-terminus, it looked like the density did not match residues from the sequence. I kept the residues as in the sequence, but as you can see from the PHENIX refined picture (below is the link) it still looks like the amino acid sequence in the crystal does not match the published protein sequence. Out of interest I refined the same file in REFMAC, and now the electron density is correct, and the sequence of the amino acids in the crystal matches the published sequence (see link for picture below). Not only that. my R/Rfree improved (16.5/19 for PHENIX, 10/18 for REFMAC). I've also refined the occupancies of the iodide, however the the output FO-FC map from PHENIX complains and the REFMAC map is fine. How can this be and what causes this? Link for the pictures: Both maps are at identical Sigma levels in both pictures. PHENIX: http://dl.dropbox.com/u/51868657/PHENIX_refined.png REFMAC: http://dl.dropbox.com/u/51868657/REFMAC_refined.png Cheers, Chris Browning - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFO4PjgUxlJ7aRr7hoRAszXAKCNmTZvCVaDUm6v3lQjp051H+ilDgCgxd1l as9CcWEseq9uEV8qMZsOfsg= =KKUr -END PGP SIGNATURE-
Re: [ccp4bb] PHENIX vs REFMAC refinement had me fooled
Hi Petr, The automatic weighting scheme in the recent Refmac version that comes with 6.2.0 is fine but there is a limit to what it can do if something is seriously wrong with the model/data/whatever. It has just worked well for me in all respects (Rw/Rf gap, maps quality, FOM, ligands, all-the-validation-parameters-you-can-think-of ) on 2.2 and 3 A resolution structures. In earlier versions (I can't recall how far back) I remember having to disable the automatic weighting and use my own values. There must be something else there in the refmac run. Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Petr Leiman [petr.lei...@epfl.ch] Sent: Thursday, December 08, 2011 9:43 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] PHENIX vs REFMAC refinement had me fooled Dear Tim, I agree with you completely. The question then becomes why does the automatic weighting scheme in refmac allow R and R-free to run away from each other by 8% in a 1.1 A resolution structure? Petr On Dec 8, 2011, at 6:50 PM, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Christopher, if your R/Rfree from Refmac5 really are 10% vs. 18%, you might simply be looking at an electron density map with strong model bias, i.e. the map shows the features of the model and not of the data. Although at 1.1A resolution this seems quite unlikely, but that's what might explain this great gap between R and Rfree. Tim On 12/08/2011 06:36 PM, Christopher Browning wrote: Dear All, Question: Has anybody ever refined the same structure using PHENIX and then tried REFMAC to see what happens? I did and I stumbled on something funny. I'm refining a structure at 1.1A resolution which was solved with Iodine phasing using PHENIX AutoSolve. Got a great map and the structure was built almost completely. I had to build a few residues myself, and using the published sequence, I started filling in the residues, but as I came nearer the N-terminus, it looked like the density did not match residues from the sequence. I kept the residues as in the sequence, but as you can see from the PHENIX refined picture (below is the link) it still looks like the amino acid sequence in the crystal does not match the published protein sequence. Out of interest I refined the same file in REFMAC, and now the electron density is correct, and the sequence of the amino acids in the crystal matches the published sequence (see link for picture below). Not only that. my R/Rfree improved (16.5/19 for PHENIX, 10/18 for REFMAC). I've also refined the occupancies of the iodide, however the the output FO-FC map from PHENIX complains and the REFMAC map is fine. How can this be and what causes this? Link for the pictures: Both maps are at identical Sigma levels in both pictures. PHENIX: http://dl.dropbox.com/u/51868657/PHENIX_refined.png REFMAC: http://dl.dropbox.com/u/51868657/REFMAC_refined.png Cheers, Chris Browning - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFO4PjgUxlJ7aRr7hoRAszXAKCNmTZvCVaDUm6v3lQjp051H+ilDgCgxd1l as9CcWEseq9uEV8qMZsOfsg= =KKUr -END PGP SIGNATURE-
Re: [ccp4bb] PHENIX vs REFMAC refinement had me fooled
My money is on the the wrong test set (as Jonathan Elegheert suggested). I have seen this several times with newbies, when the test set is created by phenix. It does it the xplor-way. When it comes to the free set, refmac defaults to 0, phenix tries to be intelligent (i.e. if 1/0 it uses 1, if more 0/1/2... it uses 0). Additionally, refmac (and I think phenix) produces Fc filled maps. So if you swap R/freeR reflections, the maps always look spectacular, as they essentially are Fcalc maps. Inspection of the logfiles should help: #of reflections free and #of reflections for refinement are reported by both programs, and IIRC, you should get a warning that your free-R-set is not sensible. One way out is to /always/ use ccp4 to assign the test-set, then both programs run fine. Otherwise you have to explicitly tell refmac to use 1 as the test reflections. HTH, Jens On Thu, 2011-12-08 at 18:36 +0100, Christopher Browning wrote: Dear All, Question: Has anybody ever refined the same structure using PHENIX and then tried REFMAC to see what happens? I did and I stumbled on something funny. I'm refining a structure at 1.1A resolution which was solved with Iodine phasing using PHENIX AutoSolve. Got a great map and the structure was built almost completely. I had to build a few residues myself, and using the published sequence, I started filling in the residues, but as I came nearer the N-terminus, it looked like the density did not match residues from the sequence. I kept the residues as in the sequence, but as you can see from the PHENIX refined picture (below is the link) it still looks like the amino acid sequence in the crystal does not match the published protein sequence. Out of interest I refined the same file in REFMAC, and now the electron density is correct, and the sequence of the amino acids in the crystal matches the published sequence (see link for picture below). Not only that. my R/Rfree improved (16.5/19 for PHENIX, 10/18 for REFMAC). I've also refined the occupancies of the iodide, however the the output FO-FC map from PHENIX complains and the REFMAC map is fine. How can this be and what causes this? Link for the pictures: Both maps are at identical Sigma levels in both pictures. PHENIX: http://dl.dropbox.com/u/51868657/PHENIX_refined.png REFMAC: http://dl.dropbox.com/u/51868657/REFMAC_refined.png Cheers, Chris Browning
Re: [ccp4bb] PHENIX vs REFMAC refinement had me fooled
Check your Rfree flag. Phenix and refmac may use different flag. Have a look into log file. If percentage of Rfree reflections is 95% then flags need to be swapped. Garib On 9 Dec 2011, at 02:50, Mark J van Raaij wrote: are you sure that you are using the original input intensities for the REFMAC map calculation (and the refinement run) and not the output ones of the (previous) run? if you are not, you may have model bias, and Rfree can be fooled, especially with NCS (if you have it). - or perhaps anisotropic B-factor refinement (if you are using it), works better in REFMAC than PHENIX? Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij On 8 Dec 2011, at 18:36, Christopher Browning wrote: Dear All, Question: Has anybody ever refined the same structure using PHENIX and then tried REFMAC to see what happens? I did and I stumbled on something funny. I'm refining a structure at 1.1A resolution which was solved with Iodine phasing using PHENIX AutoSolve. Got a great map and the structure was built almost completely. I had to build a few residues myself, and using the published sequence, I started filling in the residues, but as I came nearer the N-terminus, it looked like the density did not match residues from the sequence. I kept the residues as in the sequence, but as you can see from the PHENIX refined picture (below is the link) it still looks like the amino acid sequence in the crystal does not match the published protein sequence. Out of interest I refined the same file in REFMAC, and now the electron density is correct, and the sequence of the amino acids in the crystal matches the published sequence (see link for picture below). Not only that. my R/Rfree improved (16.5/19 for PHENIX, 10/18 for REFMAC). I've also refined the occupancies of the iodide, however the the output FO-FC map from PHENIX complains and the REFMAC map is fine. How can this be and what causes this? Link for the pictures: Both maps are at identical Sigma levels in both pictures. PHENIX: http://dl.dropbox.com/u/51868657/PHENIX_refined.png REFMAC: http://dl.dropbox.com/u/51868657/REFMAC_refined.png Cheers, Chris Browning -- Dr. Christopher Browning Post-Doctor to Prof. Petr Leiman EPFL BSP-416 1015 Lausanne Switzerland Tel: 0041 (0) 02 16 93 04 40 Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
[ccp4bb] How to assess geometry in a model?
Hi Folks I'm looking for a way to score each atom (or residue) in a model based on it's geometry. I know these scores exists because various software packages speak of outliers, even including a sigma value in some cases. So I'm looking for a simple way to get a complete list (not just outliers). Does anyone know of a package that can be made to output these scores? Thanks, Matt
Re: [ccp4bb] How to assess geometry in a model?
Hi Matt, WHAT_CHECK writes out a file called check.db that contains per-residue scores for several quality metrics. It is fairly easy to parse. Cheers, Robbie Date: Thu, 8 Dec 2011 23:08:45 -0500 From: mattw...@gmail.com Subject: [ccp4bb] How to assess geometry in a model? To: CCP4BB@JISCMAIL.AC.UK Hi Folks I'm looking for a way to score each atom (or residue) in a model based on it's geometry. I know these scores exists because various software packages speak of outliers, even including a sigma value in some cases. So I'm looking for a simple way to get a complete list (not just outliers). Does anyone know of a package that can be made to output these scores? Thanks, Matt
Re: [ccp4bb] Postdoctoral Position Available in Houston
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